The native prokaryotic CRISPR/Cas system comprises an array of short repeats with intervening variable sequences of constant length (i.e., clusters of regularly interspaced short palindromic repeats, or “CRISPR”), and CRISPR-associated (“Cas”) proteins. The RNA of the transcribed CRISPR array is processed by a subset of the Cas proteins into small guide RNAs, which generally have two components as discussed below. There are at least three different systems: Type I, Type II and Type III. The enzymes involved in the processing of the RNA into mature crRNA are different in the 3 systems. In the native prokaryotic system, the guide RNA (“gRNA”) comprises two short, non-coding RNA species referred to as CRISPR RNA (“crRNA”) and trans-acting RNA (“tracrRNA”). In an exemplary system, the gRNA forms a complex with a Cas nuclease. The gRNA:Cas nuclease complex binds a target DNA sequence having a protospacer adjacent motif (“PAM”) and a protospacer. The protospacer is complementary to a target DNA, and the gRNA is also complementary to at least a portion of the target DNA. The recognition and binding of the target DNA by the gRNA:Cas nuclease complex induces cleavage of the target DNA. The native CRISPR/Cas system functions as adaptive immune system in prokaryotes, as gRNA:Cas nuclease complexes recognize and silence exogenous genetic elements, thereby conferring resistance to exogenous genetic elements such as plasmids and phages.
It has been demonstrated that a single guide RNA (“sgRNA”) can replace the complex formed between the naturally-existing crRNA and tracrRNA. Considerations relevant to developing a gRNA, including a sgRNA, include specificity, stability, and functionality. Specificity refers to the ability of a particular gRNA:Cas nuclease complex to bind to and/or cleave a desired target, whereas little or no binding and/or cleavage of polynucleotides different (in sequence and/or location) from the desired target occurs. Thus, specificity refers to minimizing off-target effects of the gRNA:Cas nuclease complex. Stability refers to the ability of the gRNA to resist degradation by enzymes, such as nucleases, and other substances that exist in intra-cellular and extra-cellular environments. There is a need for providing gRNA, including sgRNA, having increased resistance to nuclease degradation, increased binding affinity for the target DNA, and/or reduced off-target effects while, nonetheless, having gRNA functionality.
Targeted gene editing has potential for functional gene analysis and for gene therapy applications. Approaches that introduce double-stranded breaks (DSBs) at defined target sites have improved the frequency of genome editing via homologous recombination (HR) of exogenous donor polynucleotide templates. Banga et al., Oligonucleotide-directed site-specific mutagenesis in Drosophila melanogaster. PNAS 89(5), 1735-9 (1992); Nussbaum et al., Restriction-stimulated homologous recombination of plasmids by the RecE pathway of Escherichia coli. Genetics, 130(1), 37-49 (1992); Puchta et al., Homologous recombination in plant cells is enhanced by in vivo induction of double-strand breaks into DNA by a site-specific endonuclease. Nucleic Acids Research, 21(22), 5034-40 (1993); Rouet et al., Expression of a site-specific endonuclease stimulates homologous recombination in mammalian cells. PNAS 91(13), 6064-81994 (1994); Storici et al., Chromosomal site-specific double-strand breaks are efficiently targeted for repair by oligonucleotides in yeast. PNAS 100(25), 14994-9 (2003). These approaches include engineered homing endonucleases, zinc finger nucleases (Bibikova et al., Enhancing gene targeting with designed zinc finger nucleases. Science 300(5620), 764 (2003)), transcription activator-like effector nucleases (TALENS) (Miller et al., A TALE nuclease architecture for efficient genome editing. Nature Biotechnology, 29(2), 143-82011 (2011)), and RNA-guided Cas9 nucleases (Hsu et al., Development and Applications of CRISPR-Cas9 for Genome Engineering. Cell, 157(6), 1262-1278 (2014)) to induce DSBs at defined genomic target sites. With these nuclease-mediated gene targeting approaches, a primary limitation is the low frequency with which HR occurs at the DSB relative to the frequency of non-homologous end joining (NHEJ) (Bétermier et al., Is non-homologous end-joining really an inherently error-prone process? PLoS Genetics, 10(1), e1004086 (2014)). In mammalian cells, NHEJ is the favored DSB repair modality by three orders of magnitude, with one successful HR and NHEJ event occurs per 105-107 and 102-104 treated cells, respectively (Vasquez et al., Manipulating the mammalian genome by homologous recombination. PNAS 98(15), 8403-10 (2001)).
Genome editing methods that increase the frequency of HR relative to NHEJ after a DSB is induced, and even to favor HR over NHEJ, are desired. To facilitate this, researchers have proposed guiding exogenous donor polynucleotides templates to the vicinity of the DSB by tethering the donor to a DNA aptamer that is bound by a homing endonuclease (I-SceI). Thus, the genome-editing agent carries the HR repair template to the site of the genetic modification, which will increase the local concentration of the HR template at the genomic repair site. Using this approach, HR was improved 16- and 32-fold, in human and yeast cells, respectively (Ruff, et al., Aptamer-guided gen targeting in years and human cells. Nucleic Acids Res. 42(7):e61, 2014). However, with the homing endonuclease approach, the landscape of target genomic sequences is constrained, limited by the specific 18 DNA base-pair target recognition site of the (Ice-I) endonuclease. Although it is conceivable to select new DNA aptamers to extend this strategy for use with different homing endonucleases, aptamer selection has proven to be a difficult process in practice. (Gold et al., Aptamer-Based Mutiplexed Proteomic Technology for Biomarker Discovery. PLOS ONE 5(12)e15004 2010) Additionally, engineering homing endonucleases, zinc finger nucleases and transcriptional activator-like nucleases to target novel DNA sequences remains challenging (Gaj, et al., ZFN, TALEN, and CRISPR/Cas-based methods for genome engineering. Trends in Biotech 31(7):307 (2013).
Homologous gene targeting approaches have been used to knock out endogenous genes or knock-in exogenous sequences in the chromosome. They can also be used for gene correction, and in theory, for the correction of mutations linked with monogenic diseases. However, this application is difficult, due to the low efficiency of the process. For example, Mali et al. (Science 339:823-826 (2013)) attempted gene modification in human K562 cells using CRISPR (guide RNA and Cas9 endonuclease) and a concurrently supplied single-stranded donor DNA, and observed an HDR-mediated gene modification at the AAVS1 locus at a frequency of 2.0%, whereas NHEJ-mediated targeted mutagenesis at the same locus was observed at a frequency of 38%. Li et al. (Nat Biotechnol. (8); 688-91 (2013)) attempted gene replacement in the plant Nicotiana benthamiana using CRISPR (guide RNA and Cas9 endonuclease) and a concurrently supplied double-stranded donor DNA, and observed an HDR-mediated gene replacement at a frequency of 9.0%, whereas NHEJ-mediated targeted mutagenesis was observed at a frequency of 14.2%. Li et al. also tested the possibility of enhancing HDR in Nicotiana benthamiana by triggering ectopic cell division, via co-expression of Arabidopsis CYCD3 (Cyclin D-Type 3), a master activator of the cell cycle; however, this hardly promoted the rate of HDR (up to 11.1% from 9% minus CYCD3). Kass et al. (Proc Natl Acad Sci USA. 110(14): 5564-5569 (2013)) studied HDR in primary normal somatic cell types derived from diverse lineages, and observed that mouse embryonic and adult fibroblasts as well as cells derived from mammary epithelium, ovary, and neonatal brain underwent HDR at I-SceI endonuclease-induced DSBs at frequencies of approximately 1% (0.65-1.7%). Kass and others have reported higher HDR activity when cells are in S and G2 phases of the cell cycle. Strategies to improve HDR rates have also included knocking out the antagonistic NHEJ repair mechanism. For example, Qi et al. (Genome Res 23:547-554 (2013)) reported an increase of 5-16 fold in HDR-mediated gene targeting in Arabidopsis for the ku70 mutant and 3-4 fold for the lig4 mutant. However, the overall rates were observed to be no higher than ˜5%, with most less than 1%. Furthermore, once the desired gene-targeting event was produced, the ku70 or lig4 mutations had to be crossed out of the mutant plants.
Mali, et al. US20140342458 discusses a method of altering a eukaryotic cell by transfecting the cell with a nucleic acid encoding RNA complementary to genomic DNA of the cell, transfecting the cell with a nucleic acid encoding an enzyme that interacts with the RNA and cleaves the genomic DNA in a site specific manner. The cell expresses the RNA and the enzyme, the RNA binds to complementary genomic DNA, and the enzyme cleaves the genomic DNA in a site specific manner. It states that gRNAs are flexible to sequence insertions on the 5′ and 3′ ends (as measured by retained HR inducing activity), and ssDNA donors may be tethered to gRNAs via hybridization, thus enabling coupling of genomic target cutting and immediate physical localization of repair template which can promote homologous recombination rates over error-prone non-homologous end-joining. See also Church et al. US20150031133.
Dutreix et al. US20030003547 discusses methods and compositions for gene alternation or repair based on oligonucleotide-directed triple helix formation and homologous recombination. It states that triple helix-forming oligonucleotides (TFOs) had been chosen to guide homologous donor DNA (DD) to an intended target site on genomic DNA and to position it for efficient information transfer via homologous recombination and/or gene conversion. In this approach, TFO is covalently tethered to DD through a linker. It states that the effectiveness of the TFO-DD conjugate could be explained by: (i) an increase in the local concentration of DD; and (ii) a stimulation of DNA repair by triple helix formation that could provoke recruitment of proteins involved in homologous pairing, strand exchange and/or recombination. US20030003547 provides an approach where a homing device (TFO) and a donor DNA (DD) are joined together by non-covalent interaction through an adapter oligonucleotide, which is covalently linked to TFO. It states that an oligonucleotide (natural and modified oligonucleotide (ODN), or RNA-DNA chimeric oligonucleotide (RDO)), or also a small DNA fragment (either single- or double-stranded) could be guided the target site for homologous replacement.